目的:构建容易去除标签蛋白的大肠杆菌融合蛋白表达载体.方法:PCR方法获得带有Asp-Pro-Pro前缀的重组人骨形态发生蛋白-4成熟肽(recombination human bone morphogenetic protein 4 mature peptide,rhBMP-4m)编码序列并克隆入pRSET载体...目的:构建容易去除标签蛋白的大肠杆菌融合蛋白表达载体.方法:PCR方法获得带有Asp-Pro-Pro前缀的重组人骨形态发生蛋白-4成熟肽(recombination human bone morphogenetic protein 4 mature peptide,rhBMP-4m)编码序列并克隆入pRSET载体,转化大肠杆菌后诱导表达;经免疫印迹法鉴定融合蛋白;甲酸裂解去除6His标签后细胞学实验检测其活性.结果:构建的融合表达载体序列测定结果与预期结果一致;诱导表达的DPP-rhBMP-4m融合蛋白以包涵体形式存在,占菌体总蛋白量的37%,亲和层析后目的蛋白纯度为97%;免疫印迹法鉴定显示,该融合蛋白可与相应抗血清产生特异性反应;对C2C12细胞具有良好的生物活性.结论:成功构建了利于目的蛋白纯化的pRSET-DPP-rhBMP-4m融合表达载体.展开更多
含有编码人骨形成蛋白4成熟肽(Bone morphogenetic protein 4 mature peptide,hBMP-4m)cDNA表达质粒的菌株,经50代传代,质粒不丢失,仍然稳定高表达hBMP-4m。将此菌株导入15 L NBS发酵罐中进行恒溶氧高密度发酵、诱导表达,测定发酵菌液OD...含有编码人骨形成蛋白4成熟肽(Bone morphogenetic protein 4 mature peptide,hBMP-4m)cDNA表达质粒的菌株,经50代传代,质粒不丢失,仍然稳定高表达hBMP-4m。将此菌株导入15 L NBS发酵罐中进行恒溶氧高密度发酵、诱导表达,测定发酵菌液OD600值为43.5,离心收集菌体,PAGE电泳结果hBMP-4m占细菌总蛋白量的39％。悬浮所收集的菌体、裂菌,洗涤后的包涵体中hBMP-4m占蛋白量的85％。将少量包涵体用8 mol尿素缓冲液溶解分别上SP阳离子和Q阴离子交换柱,用连续盐浓度的洗脱液洗脱蛋白,收集各蛋白峰做蛋白电泳分析,找到洗脱液的最合适盐浓度。然后将全部包涵体用8 mol尿素缓冲液溶解,上阳离子交换柱SP柱,以最佳盐浓度洗脱液洗脱,获得纯度为91％的hBMP-4m。再上阴离子交换柱Q柱,最佳盐浓度洗脱液洗脱后,所得hBMP-4m纯度为98％。展开更多
The chelate complex of Cu2＋ with 2-hydroxy-（4-methylthio）butanoate（MHA-H, the anion derived from the so-called methionine hydroxy-analogue, largely used in animal nutrition as a source of methionine, MHA） is an e...The chelate complex of Cu2＋ with 2-hydroxy-（4-methylthio）butanoate（MHA-H, the anion derived from the so-called methionine hydroxy-analogue, largely used in animal nutrition as a source of methionine, MHA） is an efficient, bioavailable trace mineral additive for animal feeding. The structure of MHA-H copper chelate was investigated by single-crystal X-ray diffraction. The crystal structure（C10H18Cu O6S2, 1, monoclinic, space group P21/c, Z = 2, a = 16.158（4）, b = 4.9733（12）, c = 9.159（2） ？, β = 104.786（4）°） exhibits that two MHA-H ligands coordinate with a CuII ion to form a square-planar environment completed to an octahedron through interaction with carbonyl oxygens of neighboring molecules, which expand to constitute a two-dimensional sheet coordination network. And a separation between the hydrophilic and lipophilic moieties like micelles was found in the packing structure. X-ray powder diffraction and thermogravimetric analysis were used to study the phase purity of the bulk sample and thermostability of complex 1, respectively.展开更多
文摘目的:构建容易去除标签蛋白的大肠杆菌融合蛋白表达载体.方法:PCR方法获得带有Asp-Pro-Pro前缀的重组人骨形态发生蛋白-4成熟肽(recombination human bone morphogenetic protein 4 mature peptide,rhBMP-4m)编码序列并克隆入pRSET载体,转化大肠杆菌后诱导表达;经免疫印迹法鉴定融合蛋白;甲酸裂解去除6His标签后细胞学实验检测其活性.结果:构建的融合表达载体序列测定结果与预期结果一致;诱导表达的DPP-rhBMP-4m融合蛋白以包涵体形式存在,占菌体总蛋白量的37%,亲和层析后目的蛋白纯度为97%;免疫印迹法鉴定显示,该融合蛋白可与相应抗血清产生特异性反应;对C2C12细胞具有良好的生物活性.结论:成功构建了利于目的蛋白纯化的pRSET-DPP-rhBMP-4m融合表达载体.
文摘含有编码人骨形成蛋白4成熟肽(Bone morphogenetic protein 4 mature peptide,hBMP-4m)cDNA表达质粒的菌株,经50代传代,质粒不丢失,仍然稳定高表达hBMP-4m。将此菌株导入15 L NBS发酵罐中进行恒溶氧高密度发酵、诱导表达,测定发酵菌液OD600值为43.5,离心收集菌体,PAGE电泳结果hBMP-4m占细菌总蛋白量的39％。悬浮所收集的菌体、裂菌,洗涤后的包涵体中hBMP-4m占蛋白量的85％。将少量包涵体用8 mol尿素缓冲液溶解分别上SP阳离子和Q阴离子交换柱,用连续盐浓度的洗脱液洗脱蛋白,收集各蛋白峰做蛋白电泳分析,找到洗脱液的最合适盐浓度。然后将全部包涵体用8 mol尿素缓冲液溶解,上阳离子交换柱SP柱,以最佳盐浓度洗脱液洗脱,获得纯度为91％的hBMP-4m。再上阴离子交换柱Q柱,最佳盐浓度洗脱液洗脱后,所得hBMP-4m纯度为98％。
基金supported by the National Natural Science Foundation of China (No. 21301139,21373158,21103135,51304158)Scientific Research Program Funded by Shaanxi Provincial Education Department (No. 2013JK0651)+1 种基金the Scientific Research Cultivating Fund (No. 201221)the Doctoral Starting Fund (No. 2014QDJ004) from Xi'an University of Science and Technology
文摘The chelate complex of Cu2＋ with 2-hydroxy-（4-methylthio）butanoate（MHA-H, the anion derived from the so-called methionine hydroxy-analogue, largely used in animal nutrition as a source of methionine, MHA） is an efficient, bioavailable trace mineral additive for animal feeding. The structure of MHA-H copper chelate was investigated by single-crystal X-ray diffraction. The crystal structure（C10H18Cu O6S2, 1, monoclinic, space group P21/c, Z = 2, a = 16.158（4）, b = 4.9733（12）, c = 9.159（2） ？, β = 104.786（4）°） exhibits that two MHA-H ligands coordinate with a CuII ion to form a square-planar environment completed to an octahedron through interaction with carbonyl oxygens of neighboring molecules, which expand to constitute a two-dimensional sheet coordination network. And a separation between the hydrophilic and lipophilic moieties like micelles was found in the packing structure. X-ray powder diffraction and thermogravimetric analysis were used to study the phase purity of the bulk sample and thermostability of complex 1, respectively.
